During the last two decades there has been a tremendous growth in experimental methods that allow for biochemical and biophysical investigations of single cells. Such methods include patch clamp recordings which can be used for measurement of transmembrane currents through a single ion channel (Hamill, et al., Pfleugers Arch. 391: 85-100, 1981); laser confocal microscopy imaging techniques that can be used to localize bioactive components in single cells and single organelles (Maiti, et al., Science 275: 530-532, 1997); use of near field optical probes for pH measurements in the cell interior; and use of ultramicroelectrodes for measurement of release of single catechol- and indol-amine-containing vesicles (Chow, et al., Proc. Natl. Acad. Sci. USA. 88: 10754-10758, 1991).
Highly specific enzymes, substrates and protein probes are available that makes it possible to detect particular components in cells. (Tsien, Annu. Rev. Biochem. (1998), 67: 509-544). The major challenge, so far, in applying such probes, drugs, and other effectors of intracellular chemistry is in introducing them into the cellular interior. Many of these agents (e.g., nanoparticles, dyes, drugs, DNAs, RNAs, proteins, peptides, and amino acids) are polar, and polar solutes are cell-impermeable and unable to pass biological membranes. Thus, the cell plasma membrane barrier acts as a physical boundary to the external solution and prevents the entrance of exogenous compounds and particles. At present, it is extremely difficult, for example, to label a cell in a cell culture with a dye, or transfect it with a gene without labelling or transfecting its adjacent neighbor. It is even more difficult to introduce polar molecules into organelles because of their size which many times is smaller than the resolution limit of a light microscope, or at least less than a few micrometers in diameter.
Microinjection techniques for single cells and single nuclei have also been described (see, e.g., Capecchi, Cell 22: 479-488, 1980, but these get increasingly difficult to implement as the size of the cell or organelle decreases. For cells and organelles measuring only a few micrometers in diameter or less, microinjection techniques become virtually impossible to use.
Cell membranes can be permeabilized by pulsed electric fields (see e.g. Zimmermann, Biochim. Biophys Acta, (1982) 694: 227-277). This technique is called electroporation. The event that leads to breakdown of the plasma membranes is the induction of a transmembrane potential over a critical value, by the applied electric field. The transmembrane potential ΔV, for a spherical cell of radius rc in a homogeneous electric field, E, is generally calculated from:ΔV=1.5rcE cos α(1−e−t/τ)  (1)                where 1.5 is a geometric factor, and α is the angle between the location at the membrane and the direction of the field. t is the pulse duration and τ is the time it takes for the membrane to achieve the induced transmembrane potential, and is described by:τ=rcCm(ρi+0.5 ρe)  (2)        where Cm is the specific membrane capacitance per unit area, and ρi and ρe are the resistivities of the intracellular and extracellular solutions, respectively.        
The electroporation technique is widely used on large populations of cells (in the order of 106 cells). Typically, cells are placed between large, plate-electrodes which generate homogeneous electric fields. Instrumentation that can be used for electroporation of a small number of cells in suspension (Kinosita and Tsong, Biochim. Biophys. Acta 554: 479-497, 1979); Chang, J. Biophys. 56: 641-652, 1989; Marszalek, et al., Biophys. J. 73: 1160-1168, 1997) and for a small number of adherent cells grown on a substratum (Zheng, Biochim. Biophys. Acta 1088: 104-110, 1991); Teruel and Meyer, Biophys. J., 73: 1785-1796, 1997) have also been described. The design of the electroporation device constructed by Marszalek, et al., 1997, supra, is based on 6 mm long 80 μm diameter platinum wires that are glued in a parallel arrangement at a fixed distance of 100 μm to a single glass micropipette. The design by Kinosita and Tsong, 1979, supra, uses fixed brass electrodes spaced with a gap distance of 2 mm. The microporator design of Teruel and Meyer, 1997, supra, relies on two platinum electrodes that are spaced with a gap distance of about 5 mm, and the electroporation chamber design by Chang uses approximately 1 mm-long platinum wires spaced at a distance of 0.4 mm.
These electroporation devices, which are optimized for usage in vitro, create electric fields that are several orders of magnitude larger than the size of a single cell which typically is 10 μm in diameter, and thus can not be used for exclusive electroporation of a single cell or a single organelle or for electroporation inside a single cell. The techniques do not offer a sufficient positional and individual control of the electrodes to select a single cell, or a small population of cells. Furthermore, these techniques are not optimized for electroporation in vivo or for electroporation of remote cells and tissue.
Electroporation devices for clinical and in vivo applications have also been designed. Examples include devices for electroporation-mediated delivery of drugs and genes to tumors (WO 96/39226) and to blood cells (U.S. Pat. No. 5,501,662) and to remote cells and tissue (U.S. Pat. No. 5,389,069). Likewise, these cannot be used to create a highly localized electric field for spatially confined electroporation.